The genetic code of prokaryotic and eukaryotic organisms has been expanded to allow the in vivo, site-specific incorporation of over 20 designer unnatural amino acids in response to the amber stop codon. This synthetic genetic code expansion is accomplished by endowing organisms with evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that direct the site-specific incorporation of an unnatural amino acid in response to an amber codon. The orthogonal aminoacyl-tRNA synthetase aminoacylates a cognate orthogonal tRNA, but no other cellular tRNAs, with an unnatural amino acid, and the orthogonal tRNA is a substrate for the orthogonal synthetase but is not substantially aminoacylated by any endogenous aminoacyl-tRNA synthetase.
The site-specific and homogeneous modification of recombinant proteins under physiological conditions is an important challenge. Cysteines and other amino acid residues in proteins can be specifically labeled by several methods(1), but site-specificity, as opposed to residue specificity(2, 3), is difficult to achieve.
Several phenylalanine derivatives can be site-specifically introduced into recombinant proteins in response to an amber codon (UAG) inserted into the corresponding gene using an evolved tyrosyl-tRNA synthetase-tRNACUA pair that is orthogonal in E. coli(4). Phenylalanine derivatives bearing alkynyl- azido- and keto-groups, that are bio-orthogonal in their chemical reactivity have been incorporated(5-8). However the introduction of aromatic amino acids at sites where aliphatic amino acids are naturally found may cause misfolding or loss of protein function; there is therefore a pressing need for methods to site-specifically incorporate aliphatic amino acids that contain bio-orthogonal chemical handles for use in protein labeling.
Use of a tRNA synthetase-tRNA pair for incorporation of novel amino acids into proteins has been performed in the art. The Methanosarcina barkeri MS Pyrrolysyl tRNA synthetase/tRNACUA (MbPyIRS/MbtRNACUA) pair is a new orthogonal pair in E. coli(9, 10). We demonstrated that the MbPyIRS/tRNACUA pair can be evolved to direct the efficient incorporation of unnatural amino acids into genetically determined sites in recombinant proteins(10) and several unnatural amino acids have now been incorporated by evolving this pair(11, 12).
Since unnatural amino acids destined for incorporation into recombinant proteins are added to cell cultures at 1-10 mM(9) it is important that they can be synthesized in gram quantities via concise, efficient syntheses. Yokoyama and coworkers recently reported the genetic incorporation of the aromatic, photoreactive lysine derivative Ne-(o-azidobenzyloxycarbonyl-lysine) using a mutant pyrrolysine synthetase/tRNA pair(11). However, the synthetic route, yield and NMR characterization of this amino acid were not reported. Very recently Chan and coworkers reported the incorporation of a direct pyrrolysine analog with an appended alkyne(14). The pyrrolysine analog was synthesized in 17% yield after 16 steps.
Shultz and Xie (Current Opinion in Chemical Biology 2005 volume 9 pages 548 to 554) disclose adding amino acids to the genetic repertoire. In the work reviewed by these authors, use is made of a naturally occurring tyrosyl amber suppressor. The active site of this tRNA synthetase was modified and then selected with the aim of excluding binding to tyrosine and with the aim of acquiring the property of binding to non-tyrosine amino acids. This work focused on binding to near neighbours of tyrosine such as tyrosine analogues. The tRNA synthetase mutants which were obtained represent the output from the sum of the selective processes used. Among other things, these required multiple rounds of selection for enrichment, followed by manual characterisation of the resulting candidates with the hope of finding specificity for a particular tyrosine analogue amongst the particular mutants obtained. It should be noted that these studies were purely confined to aromatic amino acid moieties.
Polycarpo et al (PNAS 2004 Vol 101 pages 12450-12454) disclose an animoacyl-tRNA synthetase that specifically activates pyrrolysine. In this study, it was investigated whether or not certain analogues of pyrrolysine were substrates for the pyrrolysine tRNA synthetase. Pyrrolysine is an amino acid which is not conventionally regarded as one of the 20 essential amino acids, but can be found in certain organisms such as Methanococcus bacteria. These studies used naturally occurring tRNA-tRNA synthetase pairs from Methanococcus bacteria. The experimental system was arranged as an E. coli host cell comprising a lac Z gene bearing an amber mutation. In this manner, colonies could be easily scored for translation through the amber codon by simply looking for lac Z activity by conventional X-gal staining. This study attempted to discover what analogues of pyrrolysine could be incorporated by the pyrrolysine tRNA synthetase. It was an aim to try to understand what elements of the chemical structure of pyrrolysine were recognised by the tRNA synthetase being studied. For example, carbon atoms were added or removed to pyrrolysine to create analogues, and certain bonds within the pyrrolysine molecule were rearranged to create other analogues, and the incorporation of these analogues by the pyrrolysine tRNA synthetase was studied. The most likely interpretation of the studies disclosed by Polycarpo is that some of the analogues of pyrrolysine which were used were indeed incorporated by the tRNA synthetase. Although no formal proof of incorporation in a molecular sense was presented (the data were based on functional phenotypic readout of lac Z activity), on the basis of what is disclosed it would be reasonable to conclude that some of the pyrrolysine analogues studied were indeed incorporated into proteins using their system. It should be noted that all of the chemical analogues of pyrrolysine studied were aromatic molecules.
Fekner, Li, Lee and Chan (Angew Chem Int Ed 2009 vol 48 pages 1633-1635) disclose a pyrrolysine analogue for protein click chemistry. In particular, a direct pyrrolysine analogue is disclosed, which comprises aromatic carbon groups. This aromatic compound is then incorporated into polypeptide. The techniques disclosed in this paper comprise at least about ten separate chemical synthetic steps, which is very labour intensive and time consuming. The techniques described suffer from the drawback of low yields. Overall this technique is impractical to perform routinely in the manufacture of polypeptides of interest.
Yanagisawa et al. disclose multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N-epsilon-(o-azidobenzyloxycarbonyl) lysine for site specific protein modification. It should be noted that this corresponds to a lysine-aromatic-azide arrangement, in other words the molecule incorporated into the polypeptide comprises aromatic carbon groups. Moreover, these aromatic carbon groups are photosensitive, which requires production in darkness or in extremely low light conditions. This is labour intensive and costly since numerous synthetic steps and apparatus must be operated under these conditions. This study also involves mutated tRNA synthetase.
The present invention seeks to overcome problem(s) associated with the prior art.